Method of preventing or treating pain

ABSTRACT

The present invention relates to methods of treating pain. Monocyte chemoattractant protein-1 (MCP-1) antibodies or binding fragments thereof are used to prevent or reduce behavioral hypersensitivity associated with pain.

This invention was made in the course of research sponsored by theNational Institute of Drug Abuse (Grant No. DA11276). The U.S.government may have certain rights in this invention.

BACKGROUND OF THE INVENTION

Spinal neuroimmune activation and neuroinflammation following injury areassociated with the development of behavioral hypersensitivity indifferent animal models of persistent pain states (Watkins, et al.(1995) Pain 63: 289; DeLeo and Yezierski (2001) Pain 90(1-2): 1-6;Winkelstein, et al. (2001) J. Comp. Neurol. 438: 127-139). As such,neuroimmune activation in the central nervous system (CNS) producesglial activation and upregulation of cytokines and other regulatoryproteins of the immune system (DeLeo and Yezierski (2001) supra). Spinalglial activation has been reported in rat models of neuropathyin-association with persistent behavioral hypersensitivity (Colburn, etal. (1999) Exp. Neurol. 157(2): 289-304; Sweitzer, et al. (2001) J.Pharmacol. Exp. Ther. 297: 1210-1217; Watkins, et al. (2001) Pain 93:201-205; Watkins, et al. (2001) Trends Neurosci. 24: 450-455). Spinalcytokine (i.e., IL-1, TNF, and IL-6) mRNA and protein expression arealso elevated in neuropathic injury models and exhibit a temporalrelationship with behavioral hypersensitivity (DeLeo, et al. (1996) J.Interferon Cytokine Res. 16(9): 695-700; Winkelstein, et al., (2001)supra; Sweitzer, et al. (2001) supra; Sweitzer, et al. (2001)Neuroscience 103: 529-539). Neuroinflammation, involving theinfiltration of cells into the spinal cord and DRG, occurs followingnerve injury and affects behavioral hypersensitivity (Hu and McLachlan(2002) Neuroscience 112: 23-28).

Emerging evidence in the literature indicates chemokines may modulatenociception (Boddeke (2001) Eur. J. Pharmacol. 429(1-3): 115-119).However, while neuroinflammation implicates the upregulation ofchemokines to initiate and facilitate cellular infiltration into theCNS, no study has directly investigated whether spinal chemokines areupregulated in neuropathic pain and, if so, their temporal relationshipto behavioral sensitivity with this injury.

Chemokines are a subclass of cytokines involved in the activation,recruitment and infiltration of leukocytes to an injury site. They arecategorized based on the presence and position of cysteine residues(Rollins (1997) Blood 90(3): 909-928; Luster (1998) N. Engl. J. Med.338(7): 436-445). Chemokines are synthesized locally at sites ofinflammation and establish concentration gradients which drive targetcell migration. Chemokine receptors are expressed on neurons, astrocytesand endothelial cells (Luster (1998) N. Engl. J. Med. 338(7): 436-445).Cytokines, such as IL-1 and TNF, are among the main stimuli and/ormodulators for chemokine production by macrophages, dendritic cells andendothelial cells (Luster, et al. (1998) supra; Andjelkovic, et al.(1999) Glia 28: 225-235; Luther and Cyster (2001) Nat. Immunol. 2:102-107). This is relevant to nerve injury-induced hypersensitivity asupregulation of these same cytokines plays a crucial role in the centralneuroimmune responses of persistent pain models (Watkins, et al. (1995)supra; DeLeo and Coburn (1996) supra; DeLeo, et al. (1996) In: Low BackPain: A Scientific and Clinical Overview, Weinstein and Gordon (eds),AAOS Publishers, Rosemont, Ill., p 163-185; Hashizume, et al. (2000)Spine 25: 1206-1217; Winkelstein, et al., (2001) supra). Manychemokines, including the monocyte chemoattractant proteins (MCPS),macrophage infiltrating proteins (MIPs), and regulated upon activation,normal T-cell expressed and secreted (RANTES), have all been implicatedin models of direct trauma to the CNS. Furthermore, in a peripheralexperimental allergic neuritis model, mRNA expression of chemokines hasbeen characterized using quantitative PCR methods (Fujioka, et al.(1999) J. Neurovirol. 5(1): 27-31). In parallel with the documented timecourse of symptoms in that rat neuritis model, MCP-1, MIP-1, RANTES, andIP-10 were all increased in the cauda equina. Similarly, these samechemokines (MIP-1α, MCP-1, RANTES) were rapidly upregulated in separatecentral inflammatory and mechanical contusion models (Ousman and David(2001) J. Neurosci. 21(13): 4649-4656; Miyasgishi, et al. (1997) J.Neuroimmunol. 77 (1): 17-26; McTigue, et al. (1998) J. Neurosci. Res.53(3): 368-376). In peripheral nerve injury, MCP-1 is induced in damagedtissue (Toews, et al. (1998) J. Neurosci. Res. 53(2): 260-267; Coughlan,et al. (2000) Neuroscience 97(3): 591-600).

Such a chemokine response remains complicated with regards to aperipheral injury, given both its potential benefits and ill-effects. Assuch, a balance exists between the specific neuroprotective (beneficial)and pain-promoting (harmful) responses which result as a consequence ofspinal chemokine up regulation. Spinal chemokine upregulation,specifically MCP-1, which has been demonstrated to inducemonocyte/macrophage infiltration in the spinal cord (McTigue, et al.(1998) supra), can induce macrophage infiltration in a beneficial effortto promote axonal repair and healing due to the peripheral injury. Thisupregulation, which induces macrophage and monocyte infiltration intothe spinal cord, has a beneficial effect whereby these cells promote theremoval of cellular debris and facilitate axonal regeneration (Scheidt,et al. (1986) Brain Res. 379(2): 380-384; Avellino, et al. (1995) Exp.Neurol. 136(2): 183-198; Zeev-Brann, et al. (1998) Glia 23(3): 181-190;Ma, et al. (2002) J. Neurosci. Res. 68(6): 691-702). In addition,infiltrating macrophages secrete anti-inflammatory cytokines which helpto reduce the overall central inflammatory response. Together, theseactions of promoting axonal recovery and improved cellular survival pushthis “balance” to a more reparative one, which may be beneficial inachieving a state of functional survival in the CNS. In contrast,however, these same cells also contribute deleterious effects to CNStissue influencing the balance towards a more harmful response, and cancontribute to the maintenance of a pain response. Macrophages produce ahost of neurotoxic mediators, including nitric oxide (Grzybicki, et al.(1998) Acta Neuropathol. (Berl). 95(1): 98-103; Yamanaka, et al. (1998)Neurosci. Res. 31(4): 347-350) and inflammatory cytokines, which furthercontribute to a deleterious cascade leading to secondary cellular damagein the spinal cord. Moreover, this same deleterious effect maycontribute to the maintenance of persistent pain.

Several groups of compounds are used to relieve pain, depending on theseverity and duration of the pain sensation, and on the nature of thepainful stimulus. Drugs used to relieve mild, moderate or severe painwithout causing unconsciousness are generally called analgesics. Mildanalgesics that are termed non-narcotic agents include aspirin,acetaminophen and non-steroidal anti-inflammatory drugs. Shouldnon-narcotic-based agents prove ineffective, narcotic/opioid analgesicagents such as morphine, codeine, meperidine, and the like are used totreat more severe acute or chronic forms of pain (Wingard, et al. (1991)Human Pharmacology: Molecular to Clinical, Mosby-Year Book, Inc., pp.383, 391-92).

Despite the sophistication of new analgesic agents and improvedunderstanding of the neurobiological basis of pain, current painmanagement treatment modalities involving narcotic, non-narcotic, andanxiolytic therapeutic agents have not been able to manage the sideeffect issues associated with the use of these agents. In addition, asthe dizziness, drowsiness, depression, lethargy, difficulty in beingmobile, weakness in the extremities, orthostatic hypotension,respiratory depression, gastrointestinal distress, and renal distressside effects of these agents occur, therapeutic regimens frequentlydiscontinue one agent for a less successful pain control agent. Patientsexperiencing side effects become mal- or non-compliant in taking theprescribed pain treatment regimen to manage their particular type ofpain. Finally, because of the depressive effects of these agents,healthcare personnel treat patient populations of this type more on anin-patient only setting to minimize liability issues and to monitorabuse potentials by such patients taking these particular medications.

Thus, there is a need for improved methods of treating or preventingpain.

SUMMARY OF THE INVENTION

The present invention relates to a method of preventing or treatingindividuals with pain. The method provides the administration ofantibodies or binding fragments thereof directed to monocytechemoattractant protein-1 (MCP-1).

These and other aspects of the present invention are set forth in moredetail in the following description of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The central responses specific to pain are intimately connected to thecellular responses that result from or stimulate chemokine upregulationand release. For example, chemokines induce astrocytic migration,microglial proliferation and immune cell migration, which can furtherexacerbate immune activation. The present invention provides a method ofpreventing or treating pain using antibodies to the chemokine MCP-1. TheMCP-1 antibodies or binding fragments thereof of the present inventionare useful to prevent and/or treat inflammatory pain or chronic pain.Inflammatory pain includes pain associated with an inflammatory processsuch as arthritis. Chronic pain, according to the definition proposed bythe International Association for the Study of Pain, is a pain whichpersists beyond normal tissue healing time (suggested three months:International Association for the Study of Pain, Classification ofchronic pain. Pain, 1986, Suppl. 3, S1-S226), and this implies atransition point from acute pain. Experiments, described herein,demonstrate that a MCP-1 antibody reduces behavioral sensitivityassociated with pain. The spinal chemokine MCP-1 contributes topersistent behavioral sensitivity as measured by mechanical allodynia.Moreover, the temporal response of spinal chemokine mRNA expression inrelation to mechanical allodynia is demonstrated.

In order to study the effects of potential agents for pain treatment,such as a MCP-1 antibody, a well established animal model of pain wasused. Such animal models are routinely employed in pain research inorder to both define mechanisms associated with pain in humans and totest potential treatment modalities for pain in humans. In the presentinvention, a mouse neuropathy model was used wherein all mice examinedexhibited mechanical allodynia following L5 spinal nerve transection. Arobust allodynic response was observed which was sustained over thepostoperative period, for both the 0.008 (Table 1) and 0.015 (Table 2)gram von Frey filaments. TABLE 1 Number of Paw Withdrawals ± SEMPostoperative L5 Peripheral Spinal Time (Days) Nerve Transection Sham 0 3.52 ± 0.60 2.06 ± 0.19 1  9.96 ± 0.77 6.81 ± 0.86 3 12.54 ± 0.44 5.58± 1.17 5 13.15 ± 0.56 6.25 ± 1.23 7 12.54 ± 0.89 4.50 ± 0.78 10 13.17 ±1.01 5.25 ± 1.60 14 11.33 ± 0.80 3.75 ± 1.93

TABLE 2 Number of Paw Withdrawals ± SEM Time (Days) Neuropathy ModelSham 0  2.88 ± 0.23 3.06 ± 0.23 1 12.28 ± 0.87 8.00 ± 1.04 3 12.79 ±0.76 5.92 ± 1.31 5 15.23 ± 0.69 7.88 ± 1.52 7 14.54 ± 0.90 4.75 ± 0.9810 16.50 ± 0.76 6.00 ± 2.04 14 14.00 ± 0.63 4.75 ± 1.80

These responses were significantly greater (p<0.001) than those of thecorresponding sham surgeries Responses were significantly elevated(p<0.034) over shams for all time points, with the exception of day 1 inthe 0.008 gram testing (p=0.22).

RNAse protection assays (RPA) were performed to determine the expressionof chemokines in the L5 spinal nerve transaction model. RANTES,MIP-1α/β, IP-10, and MCP-1 mRNA were constitutively expressed at lowlevels in normal spinal cord. Chemokines also included in the RPAanalysis, not detected in this injury model, were lymphotactin/XCL1,eotaxin/CCL11, MIP-2/CXCL1, and T-Cell Activation Protein-3/CCL1.Following surgery, MIP-1α/β exhibited only slight increases compared tonormal with the greatest elevation at day three. Yet, these elevationswere not significant overall or at any time point following surgery(Table 3). TABLE 3 Time (Days) RANTES MIP-1β MIP-1α IP10 MCP-1 NormalFold Induction Over Normal 1 1.00 ± 0.19 1.00 ± 0.21 1.00 ± 0.22 1.00 ±0.24 1.00 ± 0.42 Neuropathy Model Fold Induction Over Normal 1 1.38 ±0.37 1.10 ± 0.27 1.04 ± 0.32 1.18 ± 0.31 2.43 ± 0.57 3 0.75 ± 0.19 1.28± 0.42 1.49 ± 0.60 1.47 ± 0.65 4.73 ± 0.21 7 2.15 ± 0.48 0.80 ± 0.241.31 ± 0.30 2.10 ± 0.52 4.03 ± 0.72 14 1.78 ± 0.35 1.29 ± 0.38 1.35 ±0.41 1.31 ± 0.37 1.30 ± 0.43 Sham Fold Induction Over Normal 1 1.50 ±0.02 1.11 ± 0.18 1.16 ± 0.12 0.84 ± 0.04 1.76 ± 0.13 3 1.42 ± 0.55 0.60± 0.16 0.81 ± 0.18 0.72 ± 0.15 1.75 ± 0.79 7 0.95 ± 0.42 0.51 ± 0.550.85 ± 0.51 0.77 ± 0.41 2.49 ± 1.99 14 0.73 ± 0.20 0.78 ± 0.25 0.53 ±0.22 0.58 ± 0.17 0.71 ± 0.58

RANTES mRNA slowly rose over normal and sham levels and was elevatedmore than two-fold over normal levels at days 7 and 14 for theneuropathy model. While not significant at these individual time points,the overall RANTES expression in neuropathy was significantly elevatedover those levels expressed in normal spinal cord (p=0.049). However, nodifferences were detected between surgery and sham groups for RANTESunderscoring the importance of surgical exposure. Similarly, IP-10 waselevated over normal and shams at all postoperative time points with asignificant overall relationship found between the injured and shamgroups (p=0.028). The peak IP-10 levels were observed on day 7, slowlyrising after injury and then also exhibiting a decrease again on day 14.

The most dramatic changes in mRNA levels observed in this model were forMCP-1. Spinal MCP-1 mRNA levels in neuropathy were 2.5 times those ofnormal on day 1 following injury and rose to peak levels on day 3 ofnearly five times those of normal. Elevated MCP-1 levels were sustainedon day 7 and were nearly five times those of normal and twice those ofcorresponding sham levels on this day. By day 14, there was an abruptdecrease in spinal MCP-1 levels, returning to baseline. Overall, spinalMCP-1 levels following L5 spinal nerve transection were significantlygreater than normal levels (p=0.004). While not significantly differentfrom shams (p=0.18), the magnitude of these changes was robust forMCP-1, in some cases as much as 2.5 times those of sham levels. SpinalMCP-1 changes were the most immediate and the most profound of any ofthe chemokines probed. The results reported herein indicate a centralchemokine response for a peripheral injury. Furthermore, these resultsindicate a role of central immune changes contributing to the mechanismof persistent pain.

RPA analysis of rat spinal cord tissue was also conducted and showedelevated MCP-1 mRNA at day 10 following surgery. Specifically, spinalMCP-1 mRNA in nerve-injured rats was 6.76±2.41 times those levels innormal, unoperated rats; which reached statistical significance(p=0.01). The relative amount of mRNA for the MCP-1 receptor, CCchemokine receptor 2 (CCR2), using real time reversetranscriptase-polymerase chain reaction (RT-PCR) was also examined.Levels of CCR2 in rats with nerve transection were markedly elevatedover sham and normal rats. CCR2 mRNA levels increased over normal levelsas early as 4 hours following injury, reaching a peak six-fold increaseat day 4. Similar results have been found in mice lacking CCR2 (Abbadle,et al. (2003) Proc. Natl. Acad. Sci. USA 100: 7947-52). Further, thedevelopment of mechanical allodynia was totally abrogated in theseCCR2-deficient mice. Sham levels at all time points of the studiesconducted herein were not different from normal, yet injury producedsignificantly greater levels than observed for shams (p<0.006) at alltime points following 4 hours. Allodynia patterns similar to those forthe mice were observed for all rats receiving either sham or injury. Ithas been suggested that CCR2.

To examine the role of MCP-1 in modulating behavioral sensitivity in arat neuropathy model, recombinant MCP-1 and a neutralizing antibody toMCP-1 were used to enhance and neutralize MCP-1, respectively.Intrathecal administration of the MCP-1 neutralizing antibody produced adose-dependent attenuation of mechanical allodynia following nerveinjury in tests using 2 gram (Table 4) and 12 (Table 5) gram von Freyfilaments. TABLE 4 Number of Paw Withdrawals ± SEM Anti-MCP-1 Anti-MCP-1Time (Days) Vehicle-HBSS 20 μg 4 μg 0 0.63 ± 0.26 1.00 ± 0.00  0.63 ±0.18 1 9.13 ± 0.79 5.38 ± 0.65 11.25 ± 1.62 3 9.13 ± 1.53 5.63 ± 0.7810.50 ± 1.15 5 12.63 ± 0.63  4.00 ± 0.42 10.00 ± 1.34 7 8.50 ± 0.78 3.50± 0.46 13.50 ± 1.31 10 9.88 ± 0.93 2.88 ± 0.64 10.75 ± 1.85

TABLE 5 Number of Paw Withdrawals ± SEM Anti-MCP-1 Anti-MCP-1 Time(Days) Vehicle-HBSS 20 μg 4 μg 0  1.38 ± 0.38 1.13 ± 0.23  0.88 ± 0.30 110.13 ± 0.74 6.38 ± 0.71 13.38 ± 1.46 3  9.50 ± 1.81 5.00 ± 0.42 13.50 ±1.27 5 12.63 ± 1.24 5.88 ± 0.48 13.00 ± 2.00 7 10.88 ± 1.25 4.25 ± 0.7515.75 ± 1.37 10 11.25 ± 0.65 3.75 ± 0.53 13.38 ± 1.78

The low dose of 4 μg of anti-MCP-1 did not significantly alterbehavioral hypersensitivity as compared to Hanks Balanced Salt Solution(HBSS) vehicle administration. However, at the higher dose of 20 mg ofthe MCP-1 neutralizing antibody, mechanical allodynia was significantly(p<0.001) decreased for 12 gram von Frey stimulation. A similar responsewas observed for 2 gram stimulation (p<0.001). These decreases weresignificant at days 7 (p=0.003, 2 gm; p=0.002, 12 gm) and 10 (p=0.002, 2gm; p<0.001, 12 gm), despite terminating administration of theneutralizing antibody on day 5. Spinal MCP-1 protein levels wereelevated over normal for all groups nerve-injured rats (Table 6).Moreover, no side effects were observed when anti-MCP-1 antibody wasadministered at either dose. TABLE 6 Condition Fold-Increase Normal 1.0± 0.2 Neuropathy-4 μg antibody 5.16 ± 0.72 Neuropathy-20 μg antibody4.60 ± 0.86 Neuropathy-HBSS 5.02 ± 0.02Data shown as a ratio with normal values ±S.E.M.Antibody—neutralizing antibody to MCP-1.

At the 50 ng dose, no changes in mechanical allodynia were observedcompared to administration of the heat-inactivated recombinant MCP-1(rMCP-1) vehicle. Overall, there was no significant difference inallodynia between the vehicle and rMCP-1 at this dose for 12 gramtesting. Moreover, mechanical allodynia was not induced in the animalsreceiving rMCP-1 injections in the absence of neuropathy injury.

Accordingly, in a preferred embodiment MCP-1 antibodies or bindingfragments thereof are used to prevent and/or treat pain. Pain iscomprised of neurological pain such as neuropathies, polyneuropathies(e.g., diabetes, headache, and trauma), neuralgias (e.g., post-zosterianneuralgia, postherpetic neuralgia, trigeminal neuralgia, algodystrophy,and HIV-related pain); musculo-skeletal pain such as osteo-traumaticpain, arthritis, osteoarthritis, spondylarthritis as well as phantomlimb pain, back pain, vertebral pain, chipped disc surgery failure,post-surgery pain; cancer-related pain; vascular pain such as painresulting from Raynaud's syndrome, Horton's disease, arteritis, andvaricose ulcers; as well as pain associated with multiple sclerosis,Crohn's Disease, and endometriosis.

The anti-MCP-1 antibody or binding fragment thereof of the presentinvention is preferably a MCP-1 neutralizing antibody or antibodyfragment. By neutralization is intended the reduction in, or inhibitionof a biological activity of MCP-1 as measured by an in vitro or in vivotest. For example, the induction of monocyte/macrophage infiltration inthe spinal cord (McTigue, et al. (1998) J. Neurosci. Res. 53(3):368-376) may be determined.

The anti-MCP-1 antibody or binding fragment thereof of the presentinvention may in general belong to any immunoglobulin class. Thus, forexample the anti-MCP-1 antibody may be an immunoglobulin G orimmunoglobulin M antibody.

The anti-MCP-1 antibody may be of animal, for example mammalian origin,and may be for example of murine, rat or human origin. The antibody maybe a whole immunoglobulin, or a fragment thereof, for example a fragmentderived by proteolytic cleavage of a whole antibody, such as F(ab′)₂,Fab′ or Fab fragments, or fragments obtained by recombinant DNAtechniques, for example Fv fragments (as described in WO 89/02465). Theantibody fragment may optionally be a single-chain antibody fragment.Alternatively, the fragment may comprise multiple chains which arelinked together, for instance, by disulfide linkages. The fragment mayalso optionally be a multi-molecular complex. A functional antibodyfragment will typically comprise at least about 50 amino acids and moretypically will comprise at least about 200 amino acids. As used herein,an antibody also includes bispecific and chimeric antibodies.

The anti-MCP-1 antibody may be polyclonal or monoclonal antibodies.Antibodies particularly useful to practice the method of the inventioninclude recombinant anti-MCP-1 antibodies and fragments thereof, i.e.anti-MCP-1 antibodies or fragments which have been produced usingrecombinant DNA techniques.

Especially useful recombinant antibodies include those having an antigenbinding site at least part of which is derived from a differentantibody, for example those in which hypervariable or complementaritydetermining regions of one antibody have been grafted into variableframework regions of a second, different, and preferably human, antibody(as described in EP-A-239400); recombinant antibodies or fragmentswherein non-Fv sequences have been substituted by non-Fv sequences fromother, different antibodies (as described in EP-A-171496, EP-A-173494and EP-A-194276); or recombinant antibodies or fragments possessingsubstantially the structure of a natural immunoglobulin but wherein thehinge region has a different number of cysteine residues from that foundin the natural immunoglobulin, or wherein one or more cysteine residuesin a surface pocket of the recombinant antibody of fragment is in theplace of another amino acid residue present in the naturalimmunoglobulin (as described in WO89/01974 and WO89/01782,respectively).

An effective amount of MCP-1 antibody or binding fragment thereof isdefined as an amount which prevents or reduces behavioralhypersensitivity of inflammatory pain or chronic pain. Behavioralhypersensitivity of pain may include sensations that are sharp, aching,throbbing, gnawing, deep, squeezing, or colicky in nature and may bemeasured by, for example, exposure to thermal hyperalgesia or mechanicalhyperalgesia.

As provided herein, both MCP-1 and its receptor CCR2 have elevated mRNAlevels following neuropathy indicating a role in persistent pain.Therefore, as will be appreciated after reading this disclosure, one mayalternatively block the receptor of MCP-1, i.e, CCR2, to prevent ortreat pain. Examples of agents and antibodies specific for CCR2 whichmay block or inhibit the interaction between CCR2 and MCP-1 include, butare not limited to those provided by U.S. Pat. No. 6,288,103 and U.S.Pat. No. 6,458,353, respectively.

The invention is described in greater detail by the followingnon-limiting examples.

EXAMPLE 1 General Methods

Two sets of experiments were performed. Temporal spinal chemokine andchemokine receptor (CCR) expression in neuropathic pain wascharacterized and the role of spinal MCP-1 in a rodent model ofpersistent neuropathy was assessed. The chemokine and CCRcharacterization study used male C57BL/6J mice (Jackson Labs, BarHarbor, Me.), each weighing 28-30 grams at the time of surgery and maleHoltzman and Sprague-Dawley rats (Harlan, Indianapolis, Ind.), eachweighing 200-250 gram at the time of surgery. For the MCP-1 study, maleHoltzman rats (Harlan, Indianapolis, Ind.) were used, each weighing200-250 gram at the time of surgery. Animals were housed individuallywith a 12:12 hour light:dark cycle and free access to food and water.Care was taken to minimize animal discomfort and to limit the number ofanimals used.

All surgical, procedures were performed under inhalation anesthesia: 3%halothane for induction and 1.5% halothane for maintenance for micesurgeries, and 4% and 2% halothane, respectively, for rat surgeries.Animals were divided into two, surgical groups: a neuropathy grouphaving an L5 peripheral spinal nerve transection on the left side(Colburn, et al. (1997) J. Neuroimmunol. 79(2): 163-175) and a shamgroup in which the L5 nerve was exposed only. Briefly, an incision wasmade and muscle tissue retracted to expose the left transverse processwhich was then partially removed. The L5 spinal nerve was then separatedfrom the L4 nerve and transected, removing a portion of the nerve tissueto ensure complete transection. Surgical procedures for the sham groupinvolved the exposure of the L5 spinal nerve, without any manipulationor transection. Following surgery, wounds were irrigated with saline andthe fascia and skin were closed. For mice surgeries, the fascia wasclosed using 7-0 silk suture and 6-0 silk suture was used for closingthe skin. Similarly, in rat surgeries, 3-0 polyester suture was used toclose the fascia and surgical staples were used for closing the skin.All animals were recovered in room air.

Animals were tested for mechanical allodynia with von Frey filaments(Stoelting, Wood Dale, Ill.) on the ipsilateral hind paw. Mechanicalallodynia was measured as the number of hind paw withdrawals elicited bya defined non-noxious mechanical stimulus (Colburn, et al. (1997) J.supra). Animals were previously acclimated to the testing environmentand the tester and baseline measurements were determined prior tosurgery. In each testing session, animals were subjected to three roundsof ten tactile stimulations with at least ten minutes between eachstimulation. Mice were tested using 0.008 and 0.015 gram von Freyfilaments and rats were tested using two and 12 gram filaments. Allquantification of mechanical allodynia was performed by a tester blindedto the injury type and treatment.

Data were analyzed for significance with a one-way ANOVA and post hocBonferroni analysis (STATA 5.0, Stata Corporation, College Station,Tex.). Significance was defined at a p value <0.05.

EXAMPLE 2 Chemokines and CCR2 in Neuropathic Pain

Following surgery, mice (n=28 neuropathy; n=12 sham) were tested formechanical allodynia. Mechanical allodynia was measured on days 1, 3, 5,7, 10 and 14, using the 0.008 and 0.015 gram von Frey filaments asdescribed in Example 1.

Assessment of the temporal chemokine mRNA expression in the mouse spinalcord was performed using an RNAse Protection assay (RPA) technique.Lumbar spinal cord tissue was harvested on days 1, 3, 7, and 14. Tissuefrom both the neuropathy (n=7 each time point) and sham (n=3 each timepoint) groups were analyzed using RPA. Tissue from normal animals (n=6)was included in the RPA analysis for comparison. Isolation of mRNA andRPA were performed according to the manufacturer's directions(Pharmingen, San Diego, Calif.). The mCK-5 template set which detectsLtn, RANTES, Eotaxin, MIP-1α/β, MIP-2, IP-10, MCP-1, TCA-3, L32, andGAPDH was used for this analysis. The template set was synthesized intoa ³²P-labeled antisense RNA probe set, hybridized overnight with spinalRNA samples, digested with RNAse, purified, resolved on a denaturedpolyacrylamide gel and quantified by autoradiography. Two housekeepinggenes (L32, GAPDH) were included with each sample to ensure comparativeanalysis of mRNA. Image analysis was employed to compare mRNA levelsbased on band intensities for each chemokine and injury group. Theintensity of each band was measured using the public domain NIH Imagesoftware program (U.S. National Institutes of Health) and assigned anarbitrary unit based on the measured intensity levels. Image intensityfor the housekeeping genes and background levels were used to normalizechemokine measurements and compare the relative levels of mRNA acrossgroups. A time course of relative mean levels of chemokine mRNA wasdetermined for neuropathy and the corresponding shams. Chemokine levelswere normalized by the values for normal animals and reported as ratiosto (fold-increases over) normal levels.

A customized RPA chemokine probe set (Pharmingen, San Diego, Calif.) forrats was utilized in a group of Holtzman rats to confirm mRNA changes inthis species. Lumbar spinal cord tissue from L5 nerve-transected rats(n=5) was harvested on day 10 following injury and spinal mRNA leves wasanalyzed using RPA as disclosed herein. Tissue from normal animals (n=2)was also included in the RPA analysis for comparison and normalization.Isolation of mRNA and RPA were performed according to the manufacturer'sdirections (Pharmingen, San Diego, Calif.). A customized template setwas uses to probe for the following chemokines and cytokines: MCP-1,IL-1ra, caspase-1, IL-18, MIP-2, IL-10, TNF-α, L32, and GAPDH(Pharmingen, San Diego, Calif.). Image analysis was performed using theIMAGEQUANT® software, version 5.2 (MOLECULAR DYNAMICS™, Sunnyvale,Calif.) and relative levels were compared between groups and reported asa (fold-increase over) ratio to normal levels.

EXAMPLE 3 Real Time Reverse Transcriptase Transcription-Polymerase ChainReaction

Assessment of the temporal spinal cord CCR2 mRNA expression wasperformed using a Real Time Reverse Transcriptase-Polymerase ChainReaction (RT-PCR) technique. Lumbar spinal cord tissue was harvestedfrom Sprague-Dawley rats following L5 spinal nerve transection at fourhours, 1, 4, and 7 days, respectively. Tissue from both neuropathy (n=3each time point) and sham (n=2 each time point) groups were analyzedusing RT-PCR. Tissue from normal animals (n=2) was also included forcomparison. The Taqman probes/primers for CCR2 and GAPDH were designedbased on Accession Nos.:NM_(—)021866 and NM_(—)017008 sequences,respectively, using PRIMER EXPRESS™ (APPLIED BIOSYSTEMS®, Foster City,Calif.). The forward primer for CCR2 was 5′-GAGTAACTGTGTGGTTGACATGCA-3′(SEQ ID NO:1) while the reverse primer was 5′-GCAGCAGTGTGTCATTCCAAGA-3′(SEQ ID NO:2). The probe for CCR2 was 5′-TTAGACCAGGCCATGCAGGTGACAGAG-3′(SEQ ID NO:3). The forward primer for GAPDH was5′-CCCCCAATGTATCCGTTGTG-31 (SEQ ID NO:4) while the reverse primer was5′-TAGCCCAGGATGCCCTTTAGT-3′ (SEQ ID NO:5). The probe for GAPDH was5′-TGCCGCCTGGAGAAACCTGCC-3′ (SEQ ID NO:6). Probes were dually labeledwith a reporter fluorescent dye, FAM (6-carboxyfluorescein), at the 5′end and a fluorescence dye quencher, TAMRA(6-carboxytetramethyl-rhodamine), at the 3′ end. The specificity of thePCR primers was tested under conventional PCR conditions in aMASTERCYCLER® Gradient EPPENDORF thermocycler (Brinkmann InstrumentInc., Westbury, N.Y.). A single band with expected molecular size wasobserved for both CCR2 and GAPDH analyzed using 1% agarose gelelectrophoresis followed by ethidium bromide staining. Prior to thereverse transcription reaction, contaminating residual genomic DNA waseliminated by DNAseI treatment of the total RNA, using DNA-FREE™ Kit(AMBION®, Austin, Tex.). RT and real time PCR reactions were carried outusing the High Capacity cDNA Archive Kit (APPLIED BIOSYSTEMS®, FosterCity, Calif.) and the PLATINUM® Quantitative PCR Supermix-UDG Kit(INVITROGEN™, Carlsbad, Calif.). The RT reaction was carried out in a100 μL total reaction volume containing: 10 μL 10× RT buffer, 4 μL 25×dNTPs, 5 μL MULTISCRIBE™ reverse transcriptase (50 U/μL; APPLIEDBIOSYSTEMS®, Foster City, Calif.), 21 μL RNAse-free water and 10 μgtotal RNA in 50 μL. The reaction was performed at 25° C. for 10 minutes,37° C. for 120 minutes and 95° C. for 5 minutes in the MASTERCYCLER®.Real time PCR was carried out on the ICYCLER IQ™ Multicolor Real TimePCR detection system (BIO-RAD®, Hercules, Calif.), in a total reactionvolume of 25 μL containing the final concentration of 1.5 U of PLATINUM®Taq DNA polymerase, 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 3 mM MgCl₂, 200μM dGTP, dCTP and DATP, 400 μM of dUTP and 1 U Uracyl DNA glycosylase(UDG), 200 nM of forward and reverse primers, 200 nM of Taqman probe,and 5 μL of a 10-fold dilution of cDNA from the RT step. Relative CCR2levels were determined based on values of each sample normalized to theGAPDH housekeeping gene. The relative expression of the CCR2 mRNA wasexpressed as a fold-increase compared to normal values and these ratioswere compared between nerve-injured and sham-operated rats.

EXAMPLE 4 Spinal MCP-1 in Neuropathic Pain

To assess the role of spinal MCP-1 in neuropathic pain, an MCP-1neutralizing antibody and recombinant MCP-1 (rMCP-1) were eachadministered in separate neuropathy groups of L5 spinal nerve-injuredrats. An L5 spinal nerve transection was performed for all rats. Forboth agents, treatment was initiated 1 hour prior to surgery and wasadministered postoperatively on days 1, 3, and 5 via lumbar punctureunder brief inhalation anesthesia. In one subgroup of animals, the MCP-1neutralizing antibody (Pharmingen, San Diego, Calif.) was administeredat either one of two doses: a low dose of 4 μg (n=8) or a high dose of20 μg (n=8). Also, a matched group of rats (n=8) received a 20 μlinjection of Hanks Balanced Salt Solution (Gibco, Grand Island, N.Y.) asa vehicle control. In the second group of animals, either 50 ng ofrecombinant MCP-1 (Pharmingen, San Diego, Calif.) in 20 μL of sterilephosphate buffered saline (PBS)/1% BSA (n—8) or a heat inactivatedrMCP-1 vehicle (n=8) was administered. Lastly, in a separate group ofrats (n=8), 50 ng of rMCP-1 was administered in the absence of anysurgery to assess the effects of spinal MCP-1 alone on mechanicalallodynia. Mechanical allodynia was measured on days 1, 3, 5, 7 and 10postoperatively for all treatment groups.

EXAMPLE 5 Enyzme-Linked Immunosorbent Assay

Quantitative determination of MCP-1 protein was performed usingenzyme-linked immunosorbent assay (ELISA) for a subset of the treatedrats: injured (n=4 each treatment group) and normal naïve (n=2) animals.Lumbar spinal cord was harvested on day 10. All spinal cord tissue usedwas flash frozen and stored at −70° C. A 0.5 cm portion of the L4-L5region was removed from the spinal cord and homogenized in 0.20 mL ofice-cold phosphate-buffered saline (pH 7.5) containing a proteaseinhibitor tablet (Boehringer Mannheim, Indianapolis, Ind.). Samples werecentrifuged at 12,000 rpm for 30 minutes at 4° C., aliquoted and storedat −70° C. Total protein concentration was determined using the BCAassay (Pierce, Rockland, Ill.) in accordance with the manufacturer'sinstructions. The MCP-1 ELISA was performed using monoclonal mouseanti-rat McP-1 and biotinylated mouse anti-rat McP-1 as capture anddetection antibodies, respectively (Pharmingen, San Diego, Calif.).Recombinant rat MCP-1 (Pharmingen, San Diego, Calif.) was used togenerate a standard curve ranging from 1280 to 5 pg/mL. For each tissuesample, the amount of MCP-1 protein per microgram of total proteinassayed was determined. For comparison between groups, the picograms ofMCP-1 per microgram of total protein were normalized by thecorresponding values of tissue from normal animals. This normalizedratio indicates the fold-increase of MCP-1 following L5 spinal nervetransection over normal.

1. A method for preventing or treating pain comprising administering toa mammal at risk of having or having pain an effective amount of amonocyte chemoattractant protein-1 (MCP-1) antibody or binding fragmentthereof thereby respectively preventing or treating pain in the mammal.